The invention relates to polypeptide antibiotics and to the identification of genetic loci associated with expression of the antibiotics. The invention particularly describes a purified lanthionine-containing antimicrobial agent, DNA encoding the protein, and methods and compositions for treatments employing the antibiotic.
Several species of bacteria inhabit the human oral cavity; among them Streptococcus mutans is considered a major etiologic agent responsible for dental caries. Loesche (1986) Microbiol. Rev. 50:353-380. Previous studies showed a certain percentage of clinical isolates of S. mutans producing antimicrobial substances called mutacins. Caufield et al. (1985) Infect. Immun. 48:51-56; Hamada et al. (1975) Arch. Oral Biol. 20:641-648. Mutacins are active against closely related species as well as a surprisingly wide spectrum of other Gram-positive bacteria. Parrot et al. (1990) Can. J Microbiol. 36:123-130. The ability to produce mutacins, combined with lactic acid production by S. mutans may contribute to the pathogenesis of these bacteria. Kleinberg, p. 605-624, in W. A. Nolte (ed.), Oral microbiology, The C.V. Mosby Company, St. Louis. Production of mutacins by S. mutans and other oral streptococci may also play a protective role to the host against pathogens such as Group A streptococci and Streptococcus pneumoniae. In this respect, mutacins may serve as antimicrobial agents in the future.
Lantibiotics are lanthionine-containing small peptide antibiotics that are produced by gram-positive bacteria. Jung (ed.), p. 1-34, in G. Jung and H. G. Sahl (ed.), Nisin and novel lantibiotics, ESCOM Sci. Publ., Leiden; Sahl et al. (1995) Eur. J. Biochem. 230:827-853. The lantibiotics are ribosomally synthesized and post-translationally modified. The modification reactions include dehydration of serine and threonine residues and the addition of thiol groups from cycteine residues to the double bound to form lanthionines and xcex2-methyllanthiones, respectively. Some dehydrated serine or threonine may remain as such in the mature lantibiotic molecule.
Based on the secondary structures, Jung assigned lantibiotics into two classes, Type-A (linear) and Type-B (globular). Jung (ed.), p. 1-34, in G. Jung and H. G. Sahl (ed.), Nisin and novel lantibiotics, ESCOM Sci. Publ., Leiden. de Vos et al. ((1995) Molecular Microbiol. 17:427-437) and Sahl and Bierbaum (Sahl et al. (1998) Annu. Rev. Microbiol. 52:41-79) further divided each class into subgroups according to their primary peptide sequences. Thus, subgroup AI contains the nisin-like lantibiotics with nisin, subtilin, epidermin and pep5 as the most thoroughly characterized members. Allgaier et al. (1986) Eur. J. Biochem. 160:9-22; Gross et al. (1968) FEBS Lett 2:61-64; Gross et al. (1971) J Am. Chem. Soc. 93:4634-4635; Kaletta et al. (1989) Arch. Microbiol. 152:16-19; Weil et al. (1990) Eur. J. Biochem. 194:217-223. Subgroup AII consists of lacticin 481, SA-FF22, salivaricin and variacin. Hynes et al. (1993) Appl. Environ. Microbiol. 59:1969-1971; Piard et al. (1993) J. Biol. Chem. 268:16361-16368; Pridmore et al. (1996) Appl. Environ, Microbiol. 62:1799-1802; Ross et al. (1993) Appl. Environ. Microbiol. 59:2014-2021. The genes responsible for the biosynthesis of the lantibiotics are organized in operon-like structures. The biosynthesis locus of all members in the subgroup AI lantibiotics consists of lanA, the structural gene for the lantibiotic; lanB and lanC, the modifying enzyme genes for post-translational modification of the preprolantibiotic; lanP, the protease gene for processing of the prelantibiotic; and lanT, the ABC transporter for secretion of the lantibiotic. In addition, epidermin and gallidennin have an extra gene, lanD, which is responsible for the C-terminal oxidative decarboxylation of the lantibiotic. Kupke et al. (1994) J. Biol. Chem. 269:5653-5659; Kupke et al. (1995) J. Biol Chem. 270:11282-89. In comparison, subgroup AII lantibiotics have simpler genomic organizations. In subgroup AII, lanB and lanC are combined into one gene, lanM, and lanP and lanT are combined into lanT. Chen et al. (1999) Appl. Environ. Microbiol. 65:1356-1360; Qi et al. (1999) Appl Environ. Microbiol 65:652-658; Rince et al. (1994) Appl. Environ. Microbiol. 60:1652-1657. All lantibiotic loci also contain a set of immunity genes, which are responsible for self-protection of the producer strains. Saris et al. (1996) Antonie van Leewenhoek 69:151-159. Moreover, the expression of the lantibiotic genes is usually regulated either by a single transcription regulator (Peschel et al. (1993) Mol. Microbiol. 9:31-39; Qi et al. (1999) Appl. Environ. Microbiol. 65:652-658) or by a two-component signal transduction system (de Ruyter et al. (1996) J. Bacteriol. 178:3434-3439; Klein et al. (1993) Appl. Environ. Microbiol. 59:296-303; Kuipers et al. (1995) J. Biol. Chem. 270:27295-27304).
Previously, the isolation, biochemical and genetic characterizations of mutacin II, produced by a group II strain of the oral bacteria Streptococcus mutans was reported. Chen et al. (1999) Appl Environ. Microbiol. 65:1356-1360; Novak et al. (1994) J. Bacteriol. 176:4316-4320; Novak et al. (1996) Anal Biochem. 236:358-360; Qi et al. (1999) Appl. Environ. Microbiol. 65:652-658. Mutacin II belongs to subgroup AII in the lantibiotic family. Recently, the isolation and genetic characterization of mutacin III from the group III S. mutans strain UA787 was reported. Qi et al. (1999) Appl. Environ. Microbiol. 65:3880-3887. The mature mutacin III is twenty-two amino acids in size, and shows striking similarity with another lantibiotic, epidermin, produced by Staphylococcus epidermidis. Allgaier et al. (1986) Eur. J. Biochem. 160:9-22. The mutacin III biosynthesis gene locus consists of eight genes in the order of mutR, -A, -Axe2x80x2; -B, -C, -D, -P, and T. The genomic organization and primary sequence of mutacin III places it in subgroup AI with epidermin and galliderniin as its closest neighbors. Applicants disclosed herein the biochemical and genetic characterization of mutacin I. Comparison of the biosynthesis genes between mutacin I and mutacin III reveal striking similarities as well as important differences.
The cloning and sequencing of the novel mutacin I biosynthetic genes by using information from the conserved sequence derived from several other lantibiotics, and the isolation and purification of mutacin I is disclosed herein and provides a novel group of antibiotics which can be utilized as anti-microbial agents against, for example, presently antibiotic resistant microorganisms.
According to the present invention, an isolated and purified DNA sequence which encodes a lantibiotic, mutacin I, is disclosed. The nucleic acid sequence is set forth in SEQ ID No: 1 and the amino acid sequence is set forth in SEQ ID No: 2. Also disclosed are pharmaceutical compositions containing mutacin I and methods for their use.